Throughout this specification, various publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this specification in order to more fully describe the state of the art to which this invention pertains.
This application is directed towards novel expression plasmids for expression of proteins under control of an E. coli osmB promoter.
A number of E. coli promoters are known and have been applied to obtain bacterial expression of proteins. Commonly used promoters are the .lambda. bacteriophage promoters .lambda.P.sub.L or .lambda.P.sub.R which are regulated by the thermolabile repressor cI.sup.857. Although highly efficient in driving expression of cloned genes, the system requires the presence, in the host or in the plasmid, of the thermolabile repressor cI.sup.857. Such systems are induced by incubating the cell culture at 42.degree. C. The elevated temperature of induction may enhance protein misfolding and may lead to the formation of insoluble protein aggregates in the form of inclusion bodies.
Other promoters such as the tac, lac or trp promoters require induction by addition of expensive chemical inducers to the medium. With the exception of the tac expression system, most of the other promoters are weaker and less efficient than .lambda.P.sub.L.
Another known promoter is the E. coli deo promoter. This is a constitutive promoter that shows variable promotion in the absence of glucose.
The osmB gene has been described by Jung et al. (1989) (1) and Jung et al. (1990) (2). The osmB gene, located at position 28 min on the E. coli chromosome, encodes an outer membrane lipoprotein containing 49 amino acids. A signal peptide directing the protein to the outer membrane is composed of 23 amino acids. The signal peptide and the osmB mature gene product are similar in many respects to the lpp gene product of E. coli (1). Expression of these peptides is promoted by both osmotic pressure (hyperosmolarity) and the ageing process, i.e. by entering the stationary phase of growth. Both events control expression at the level of transcription. Two transcription initiation sites have been identified by RNase protection of in-vivo message. The two initiation sites are designated P1 and P2. The site P2 is located 150 base pairs downstream from P1 and is the primary site of regulation that responds to either elevated osmolarity or to growth phase signal. The P1 promoter is activated only when both osmotic and growth phase signals are present simultaneously. A 7 base pair sequence upstream from P2 has been identified as the cis-acting regulatory element essential for the osmotic stimulation of osmB expression. Expression of osmB at the stationary phase is triggered directly from P2 (2). The nucleotide sequence of the promoter region of osmB is presented in FIG. 1 as described by Jung et al. (1).
Applicant has succeeded in producing recombinant plasmids which contain the osmB promoter. These plasmids are novel expression systems, not previously disclosed, and they can be used to produce high levels of a wide variety of recombinant polypeptides under control of the osmB promoter.